Method and apparatus for electrophoretic focusing

ABSTRACT

An apparatus and method is provided for obtaining a preparative-scale, free-fluid electrophoretic separator with high resolution as well as an analytical capability commensurate with capillary zone electrophoresis. The electrophoretic focusing apparatus and method of the present invention combines features of electrophoresis and isoelectric focusing to accomplish large scale purifications and fractionations that have not previously been possible, and features a separation chamber bounded by glass-coated porous metal screens, a plurality of purge chambers, a plurality of electrode chambers, and a plurality of pump means. The separation device of the invention is capable of high speed of separation and short residency of sample through the use of high voltage gradients which are produced by relatively low voltages applied across the narrow chamber dimensions. The present invention is also highly flexible, with operation in either a constant electric field, continuous flow mode or in a linearly varying electric field, batch mode, and both modes permit scanning of the sample fraction content and display in a conventional histogram format. The present apparatus and method thus achieves high resolution of separation in an analytical or a preparative mode through a practically unlimited scale-up potential, and controls the adverse effects of Joule heating and electrohydrodynamics on the electrophoretic separation procedure.

1. This application is a divisional application of U.S. application Ser.No. 09/277,944 filed Mar. 29, 1999.

FIELD OF THE INVENTION

2. The invention relates in general to an apparatus and method forachieving electrophoretic focusing, and in particular to an apparatusfor achieving electrophoretic separation and purification which ischaracterized by a separation chamber formed between two precision-pore,glass-coated metal screens and which also includes inlet and outletports, a plurality of purge chambers for extracting extraneous fractionsand for providing thermal cooling, a plurality of electrode chambers toprovide a transverse electric field in the separation chamber, andpumping means for pumping sample, carrier buffer and electrode rinsebuffer through the apparatus, and a method of employing this apparatusto achieve separation and collection of a desired component from abiological or chemical sample.

BACKGROUND OF THE INVENTION

3. There are two electrokinetic methods that have had success separatingbiological materials, namely, zone electrophoresis and isoelectricfocusing. Electrophoresis is the movement of suspended or dissolvedcharged particles in response to an applied electric field. The rate ofmotion depends upon the charge, size and shape of the particles andspecific properties of the solvent buffer and its container. In zoneelectrophoresis, the components in a short sample zone are separated bythe action of the electric field. The injection of a narrow, uniformzone and the absence of dispersive fluid flows are necessary conditionsfor successful operation. Significant sources of dispersion are: 1)uneven (parabolic) flows; 2) electrohydrodynamic flows; 3) moleculardiffusion; 4) thermal convection; 5) sedimentation; 6) thermally inducedsample mobility variations; and 7) electroosmosis.

4. In continuous zone electrophoresis (CFE), the electrolyte solutionflows in a direction perpendicular to the electric field and the mixtureto be separated is inserted continuously into the flowing solution.Components of the mixture are deflected according to theirelectrophoretic mobilities and can be collected continuously after theirmigration. Svensson and Brattsten were the first to report a method forcarrying out electrophoresis continuously. They used a lateral electricfield in a narrow plexiglas box packed with glass powder as ananti-convective medium. Durrum modified the above configuration byreplacing the glass-filled box with a filter paper curtain, hanging in afree vapor space. While both of these methods demonstrated continuouselectrophoresis, they both used a stabilizing medium. Anti-convectivemedia cause many problems such as reduction of the flow capacity bytheir presence, electroosmosis in the interstices, adsorption of thesample and “packing or eddy diffusion”. Efforts were then made to docontinuous electrophoresis in a free fluid. Bier in 1957 reported thefirst continuous flow electrophoresis device which could separate twoprotein solutions by adjusting the buffer pH relative to the isoelectricpoint of one of the solutions. The device which he described as“continuous free-boundary flow electrophoresis” did not take place in asingle rectangular chamber and did not produce a separation of highpurity. Dobry and Finn (U.S. Pat. No. 3,149,060) were the first toreport continuous flow free fluid electrophoresis in a rectangularchamber with a cross-section of low aspect ratio, hence providing littleresistance to thermal convective flow disturbances. This configurationwas limited to very low electric fields and required the use of bufferthickening agents to suppress convective eddies. Philpot described acontinuous flow electrophoresis system with the electric field appliedacross (perpendicular to) a thin film of liquid. He later wrapped histhin film geometry into a thin annulus surrounded by two concentriccylinders (electrodes). The outer cylinder rotated to provide astabilizing velocity gradient.

5. Although a large throughput, 10 g/hr, was accomplished by theBiostream, its resolution was poor. This was followed by forced flowelectrophoresis devised by Bier for the large scale purification of asingle component in a mixture. Giddings extended this development withfield flow fractionation wherein an electric field has been just oneexample of the force field deflecting the sample across the narrowplane. The need for flat, uniform surfaces that also serve to isolatethe electrode arrays have slowed this development. Mel in 1959 reportedthe first use of a high aspect ratio rectangular separation chamberusing a lateral electric field. The “thin” chamber of 0.7 cm thicknessprovided the necessary wall interaction to suppress thermal convectiveflows to the extent that a less viscous free flow buffer could be used.This design served as the impetus for the development of theconventional CFE machines of the 60's and 70's with their chambercross-section of high aspect ratio and laterally directed electricfields. During this time frame, Hannig and his co-workers developed CFEby making the chamber cross-sections even thinner, approaching 0.25 cmfor some designs. Unfortunately, the gains made in suppressing thermalconvection were wiped out by electrohydrodynamic interaction withintrinsic chamber fluid flows to cause crescent-shaped distortions.Nevertheless, a variety of CFE instruments were manufactured accordingto the designs of Hannig (in Germany) and Strickler (in the US) (U.S.Pat. No. 3,412,008) and several hundred instruments were used inlaboratories around the world. Rhodes and Snyder subsequently devised atechnique to minimize these flow distortions (U.S. Pat. No. 4,752,372).

6. The concept of counterflow to oppose the electrophoretic migrationwas first described to the inventors by Griffin and McCreight as a meansto attenuate the crescent shaped distortion in CFE chambers. Richmansubsequently patented a similar counter-flow method where axial bands ofelectroosmotic coatings of varying zeta potential would “straighten”distorted sample bands (U.S. Pat. No. 4,309,268). The method wasimpractical because most coatings change with time and there exists nospectrum of coatings with respect to zeta potential. A more practicalapproach that did not use counter-flow was suggested by Stricklerwherein the CFE was divided into two vertical compartments, each with adifferent wall coating, so that the combined electroosmotic flow wouldyield a more coherent sample band. Subsequently, Ivory used counter-flowto increase sample residence time in a recycling CFE. Egen, et al. havealso devised a counterflow gradient focusing method (U.S. Pat. No.5,336,387).

7. While the crescent phenomenon was long known to cause untenablesample stream distortion in CFE instruments, it was not until 1989 thatRhodes and Snyder showed that electrohydrodynamics transforms initiallycircular sample streams into ribbons that initiate the crescent shapeddistortions. The operation of CFE devices was labor intensive andunreliable due to contamination of the closely spaced chamber walls andthe resultant electroosmotic flow variations through the chamber.

8. Isoelectric focusing (IEF) is an electrophoretic technique that addsa pH gradient to the buffer solution and together with the electricfield focuses most biological materials that are amphoteric. Amphotericbiomaterials such as proteins, peptides, nucleic acids, viruses, andsome living cells are positively charged in acidic media and negativelycharged in basic media. During IEF, these materials migrate in thepre-established pH gradient to their isoelectric point where they haveno net charge and form stable, narrow zones. Isoelectric focusing yieldssuch high resolution bands because any amphoteric biomaterial whichmoves away from its isoelectric point due to diffusion or fluid movementwill be returned by the combined action of the pH gradient and electricfield. The focusing process thus purifies and concentrates sample intobands that are relatively stable. This is a powerful concept that hasyielded some of the highest resolution separations, especially whencoupled with electrophoresis in two-dimensional gels. Unfortunatelythere are drawbacks to IEF that have limited its applications. The rateof electrophoretic migration of each charged species decreasesprogressively as it approaches its isoelectric point and long residencetimes are required for high resolution. Proteins have reduced solubilityat their isoelectric point although precipitation of the concentratedbands can be minimized by addition of detergent. Additional problemsrelate to the commercial amphoteric solutions, including: 1) difficultyof extracting the separated proteins, peptides, etc., from theamphoteric solutions because of their similar physical properties andinteractions; 2) chemical toxicity; 3) handling problems; and 4) cost.

9. IEF had its practical beginning in the mid-1950's when Kolin firstdemonstrated the concept of focusing ions in a pH gradient by placing amolecular sample between an acidic and a basic buffer and applying anelectric field. Although the constituents focused rapidly, the gradientsoon deteriorated due to the concurrent electrophoretic migration of allof the buffering ions. The synthesis of stable carrier ampholytes byVesterberg and their successful commercial development led to broad usein gels or other restrictive media to suppress electroosmosis andthermal convection during analytical separations.

10. The high resolution achieved by IEF encouraged many attempts todevelop a preparative version of the process. This proved to be muchmore difficult for IEF than zone electrophoresis because of the variablefluid properties and sample characteristics within the chamber leadingto changing values of electroosmosis and thermal convection during theseparation. Various CFE devices were modified to run with an amphotericmixture instead of buffer but the problems (long focusing time requiringa slow flow through the chamber, pH drift toward the cathode, reducedvoltage/current levels for acceptable heating and convection) becameinsurmountable. A. J. P. Martin described a means of performinglarge-scale isoelectric focusing by connecting a number of separationchamber in series via membranes. By circulating the fluids in eachcompartment through external coolers, Martin claimed that the removal ofheat had been solved. Since the only pH shift occurred across themembranes, the pH gradient was quite steep between chambers. Bierfurther developed the external cooling system, added sensors anddemonstrated the improved focusing with recycling (U.S. Pat. No.4,362,612). Bier added a stabilizing assembly rotation to the membranesegmentation and a novel collection system (U.S. Pat No. 4,588,492)which led to the Roto-Phor from Bio-Rad (Hercules, Calif.). Righetti hasalso extended the multi-compartment concept by using membranes, cast andpolymerized with the desired amphoteric molecules inside, to establishthe pH gradient rather than preparing a constant pH in each compartment.The Iso-Prime system (Hoefer Instruments, San Francisco, Calif.) isbased upon a stack of membranes with buffer between them. The pHgradient develops rapidly and the proteins move through the membranesuntil they reach the cell with the pH equal to their isoelectric point.Although the membranes stabilize the focusing process, they becomeclogged if the protein precipitates in them.

11. Thus, prior methods of isoelectric focusing have suffered from themany drawbacks outlined above, and have also been hindered by problemsduring the transition from an analytical system to a preparative systemthat have limited its intended use. It is thus highly desirable todevelop a focusing system for separating biological molecules and othercomponents in a mixture which is able to avoid all of the problems ofthe prior art and which can achieve high resolution of separation in ananalytical or a preparative mode through a practically unlimitedscale-up potential. It is also highly desirable to develop anelectrophoretic focusing system which can control the adverse effects ofJoule heating and electrohydrodynamics on the electrophoretic separationprocedure.

SUMMARY OF THE INVENTION

12. It is an object of the present invention to provide apreparative-scale free-fluid electrophoretic separator with highresolution as well as an analytical capability commensurate withcapillary zone electrophoresis. The particular mode of high-resolutionseparation as provided by the present invention, which is referred to aselectrophoretic focusing, combines features of electrophoresis andisoelectric focusing to accomplish large scale purifications andfractionations that have not been possible before now.

13. It is another object of the present invention to develop aseparation device capable of high speed and short residency through theuse of high voltage gradients. These high voltage gradients are producedby relatively low voltages applied across the narrow chamber dimensions.Another object is flexibility with operation in either a constantelectric field, continuous flow mode or in a linearly varying electricfield batch mode. Both modes permit scanning of the sample fractioncontent and display in a conventional histogram format. The goal of highresolution of separation can be achieved through the use of the presentinvention in an analytical or a preparative mode through a practicallyunlimited scale-up potential. A further goal is to control the adverseeffects of Joule heating and electrohydrodynamics.

14. These and other objects and benefits are achieved by the use of thepresent invention which provides a number of innovations and insightswith regard to fundamental fluid and thermal geometries and operations.The focusing is accomplished with a minimum of sample migration whichleads to a higher resolution in a shorter time. Adiabatic thermalconditions in the lateral (scale-up) dimension permit a large increasein throughput at no apparent loss of resolution. Active cooling limitsthe maximum chamber temperature and its relationship to the chamberorientation and buffer fluid transport is such as to limit thermalconvection. Porous, rigid screens permit a controlled focusingcross-flow which balances the electrophoretically-driven samplevelocity.

BRIEF DESCRIPTION OF THE DRAWINGS

15.FIG. 1 is a side schematic view of the separation chamber of thepresent invention, taken in the axial and transverse directions.

16.FIG. 2 is a side schematic view of the separation chamber of thepresent invention, taken in the lateral and transverse directions.

17.FIG. 3 is a schematic view of one of the injector/collectors of thepresent invention.

18.FIG. 4 is a side schematic view of an alternative embodiment of theseparation chamber of the present invention, taken in the axial andtransverse directions.

19.FIG. 5 is a side schematic view of an alternative embodiment of theseparation chamber of the present invention, taken in the lateral andtransverse directions.

20.FIG. 6 is a schematic view of an alternative embodiment of one of theinjector/collectors of the present invention.

21.FIG. 7 is a side schematic view of a modified form of the separationchamber of the present invention.

22.FIG. 8 is a graphic representation of the electric field lines of anumerical solution of equations relating to the electric fieldcomponents of the apparatus of the present invention.

23.FIG. 9 is a side schematic view of an alternative embodiment of theseparation chamber of the present invention, taken in the axial andtransverse directions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

24. In accordance with the present invention, there is provided anelectrophoretic focusing apparatus and method which is useful inachieving the separation and purification of particular components of amixture of biological or chemical materials. The general purpose of theinvention is a continuous processing system that separates and purifiesany soluble or microparticulate sample that acquires a surface electriccharge when immersed in a polar (e.g. aqueous) fluid environment. Itcombines the best features of electrophoresis and isoelectric focusingin a novel device that incorporates a combination of transverse electricfield gradient and buffer flow field to focus and collect any selectedbiological component. Although the high resolution achievable byfocusing is familiar to isoelectric focusing, electrophoretic focusingavoids many of its problems, such as the need for complex buffers andthe long times required for the molecules to reach their isoelectricpoint. This new concept incorporates a large-gap chamber and control ofall sources of sample dispersion. The design of the electrophoreticfocusing chamber combined with the orientation and magnitude of theelectric fields and buffer flows are planned to eliminate sampledispersion. The large gap will keep sample away from the walls as wellas increase its throughput.

25. Many research and applications tasks with biological materialsrequire a large source of highly purified biologically active molecules.The diverse supply of materials for biotechnology ranging from plants togenetically derived sources are placing increased demands on separationand purification. Existing preparative separation techniques yieldproducts with a variety of impurities that can be measured analyticallybut not removed. Analytical techniques have been perfected in recentyears but attempts to scale these techniques into larger production haverelied on generally increasing the physical dimensions instead ofinvestigating a new technique. It is an advantage of the focusing deviceof the present invention that it will be able to purify biologicalmaterials in amounts and to purity levels above those now obtainable.

26. The principle of electrophoretic focusing utilized in conjunctionwith the present invention consists of opposing the electrophoreticsample velocity with a uniform fluid flow transverse to the direction ofcarrier flow through the chamber. Basically, this is the concept ofcounterflow which is discussed above. The uniqueness of this inventionis how this principle is used in conjunction with both constant andvarying voltage fields to achieve a novel and powerful method ofcontinuous sample separation. This result is achieved by using acombination of electrode arrays and insulated screens to provide theelectric field gradient and uniform transverse flow necessary forfocusing.

27. If the electric field is configured in the transverse direction(instead of the lateral direction as with CFE), electroosmotic flowbecomes negligible and the viscous parabolic flow is orthogonal to themigration direction and hence also ceases to be a factor. Since thetransverse migration is now in the narrow chamber dimension, the sampleresidence time is quite short and resolution will suffer. However, if across-flow is used, the sample will be held in the chamber by thecross-flow, thus improving the resolution by some calculatable amount.This solution to the problems of CFE has been considered by pastinventors but the problem of the area electrode/chamber broad wall haskept this idea from realization. As the details of the invention show,this problem is solved by a unique utilization of micro-pore, thin,rigid, insulating screens.

28. The electrophoretic separator of the present invention is primarilycharacterized by a separation chamber formed between two precision-poreglass-coated metal screens. The perforations permit transverse fluidflow through the chamber to effect a separation of one or multiplespecies and also to provide cooling in the chamber interior. This uniqueuse of cross-flow focuses one sample fraction continuously in thechamber when using a constant electric field to oppose the cross-flow oralternatively focuses a mobility spectrum of the sample in the chamberwhen using a linearly varying electric field. Since the separation iscarried out in the direction transverse to the carrier buffer flow, thefocusing is accomplished with a minimum of sample migration which leadsto a higher resolution in a shorter time. The relatively shorttransverse dimension allows the use of a high voltage gradient derivedfrom a low source voltage. When using low voltage gradients (to 100V/cm), the sample is injected and collected in singular or multipleports so that the chamber is only partially filled with sample. Atvoltage gradients from 100 V/cm to 1000 V/cm, the chamber thickness isgreatly reduced so that the sample fills the entire chamber. Thisconfiguration provides a homogeneous medium between the chamber wallsand eliminates conductivity gradients which produce destructivecirculatory flows through Joule heating and electrohydrodynamics.

29. Another problem with CFE is the method of sample collection. Theseparated fractions must be collected by a finite number of collectionports which ultimately limit resolution. Collection for a batch process,such as chromatography or capillary electrophoresis (CE), poses no suchproblem as each separate fraction can be individually collected over avariable time interval rather than a limited fixed distance intervalbetween each adjacent collection port as with CFE. This invention cancollect fractions as a function of time by varying the crossflowvelocity to produce a histogram similar to that obtained fromchromatography or capillary electrophoresis.

30. The present invention is capable of operation as a true focusingdevice in the same manner as IEF except that no pH gradient is utilized.A linear varying electric field gradient is produced by an electrodearray with a parabolic voltage distribution impressed on the array. Thissystem uses a homogeneous conventional buffer system in contrast to thecomplex, multicomponent systems needed for conventional IEF. Sinceelectrophoretic focusing can be done with either a constant voltagegradient or with a linearly varying voltage gradient, the two respectiveconfigurations of the present invention will be described separately.

31. I. Constant Voltage Gradient Configuration

32. FIGS. 1 and 2 show the total chamber (or simply, the chamber) andthe different flow regions. The chamber is comprised of a plurality offlow regions or sub-chambers, such as the five elements 1, 4, 5, 26, and27 shown in FIG. 1. In the preferred embodiment, the separation chamber1 is bounded by two fine mesh glass-coated metal screens, 2 and 3.Carrier buffer enters the separation chamber through the inlet manifold15 and port 21. The buffer flows through the chamber as shown with acenter plane velocity of U_(c) and exits the separation chamber via theexit port 24 and manifold 18. Sample is injected in the form of a laminathrough the injection port 40 in the injector 12 shown in detail in FIG.3. The injector is made of glass-coated metal tubing and located in thechamber as FIG. 2 shows. Note that the sample lamina does not fill theentire separation chamber width (y-direction). Separated sample iscollected through slit 40 in the collector 13. Sample can still beinjected as a lamina through an injection port 40 without requiring theinjector 12 by placing the injector port 40 in the center of theseparation chamber entrance wall. Carrier buffer will now enter theseparation chamber through flanking buffer entry ports 21 and associatedmanifolds 15 located in each edge of the entrance wall. The convergingbuffer flows will reduce the thickness of the sample lamina. In asimilar manner, separated sample can be collected through a collectionport 40 located in the center of the separation chamber exit wall withthe buffer exiting the chamber through two flanking ports 24 andmanifolds 18 located in each exit wall, as illustrated in FIG. 9.

33. As best shown in FIG. 1, adjacent co-directed flows U_(p) take placein the purge chambers 4 and 5. These flows enter through inlet manifolds14, 16 and inlet ports 20, 22. The flows exit through the manifolds 17,19 and ports 23, 25. An electric field E_(o) is impressed in the chamberby electrodes 10 and 11. These electrodes are located respectively inelectrode chambers 26 and 27. Rinse flows of center plane velocity U_(e)take place in these chambers through manifolds 28, 30 and ports 29, 31.Membranes 8, 9 isolate the electrode chambers to contain electrolysisgas and products which are swept away by the electrode rinse flow U_(c).The membranes 8, 9 are rigidized by the glass-coated metal screens 6,7.This is accomplished by keeping the pressure in the electrode chambersgreater than that in the rest of the chamber. This allows electriccurrent to flow through the membranes while keeping electrolysisproducts and flow disturbances confined to the electrode chambers. Afocusing flow velocity V_(o) is established in the separation chamber 1by a fluid flow which enters through inlet ports 35 and manifolds 37located in the purge chamber 5. This flow exits the purge chamber 4through exit ports 34 and manifolds 36. This flow V_(O) is rendereduniform in the separation chamber by virtue of the small pores inscreens 2 and 3 (e.g., roughly 0.006 inch diameter) and the large purgechamber volume which serves as a flow manifold. Two evacuated (38) glasswalls 39 bound the chamber in the lateral dimension as FIG. 2 shows.

34. It is important to eliminate lateral temperature gradients in theseparation chamber 1. The laterally directed electrode rinse flows U_(e)could possibly cause lateral temperature gradients to exist in theseparation chamber 1. For this reason, the rinse flows in the electrodechambers are oppositely directed as FIG. 2 shows. To further attenuatethese lateral temperature gradients, baffles can be placed laterally inthe electrode chambers and spaced in the axial direction. Flows inalternating directions can then be impressed by an appropriate manifoldsystem. These lateral temperature gradients originating in the electrodechambers are further averaged out by the purge flow U_(p) taking placein the purge chambers 4 and 5.

35. If the upper electrode 10 is negative and the bottom electrode 11 ispositive, an electric field E_(O) exists in the separation chamber 1which will cause a negatively charged sample to migrate down (transversedirection) under the influence of the electric field E_(O) against theuniform transverse focusing flow velocity V_(O). Consider a samplefraction of electrophoretic mobility, μ_(i)=V_(O)/E_(O) that has beeninjected through the port 12 located on the separation chamber centerplane. The sample fraction μ_(i) will remain at the center plane of theseparation chamber 1 and move through it with a carrier buffer velocityV_(c) and be collected at the exit port 13. All other sample (mobilitydifferent than μ_(i)) will exit either through port 24 in the separationchamber or through ports 23 and 25 in the purge chambers. A samplefraction scan can be made by varying V_(O). The effluent from collectionport 13 enters an ultraviolet detector and is displayed as aconventional histogram.

36. Thus, by varying the transverse focusing flow U_(o) against aconstant electric field E_(o), a scan of the fraction content of asample can be made. This type of scan of a sample is unique in aseparation device since the peak histogram is a function of the timerate of change of the focusing velocity V_(o) and is given byμ_(i)=V_(o)/E_(o). The time rate of change V_(o) is controlled by aprecision computer controlled pump. This allows real time control of theseparation process. Continuous sample collection can be made by stoppingthe scan at a peak of interest, or made after the complete scan has beenmade by recovering the transverse velocity V_(o) corresponding to a peakof interest.

37. The peak values are detected by a liquid chromatography flow celland detector system and fed back into the computer to achieve afeed-back control system. Cooling of the electrode chambers 26, 27 isprovided by the electrode rinse flow while the purge flow U_(p) providescooling for the rest of the chamber. The flow velocity U_(p) in thepurge chambers 4, 5 may be up to ten times that in the separationchamber 1 in order to accomplish this purpose. The pore size of thescreens 2, 3 is small (presently 0.006 inch hole, 34% open area) andthickness 20 gauge. While the small holes will dampen disturbance flowsbetween the separation chamber 1 and the purge chambers 4, 5, it isadvisable to consider pressure drops in the separation and purgechambers so that b_(p) ²/b_(c) ²=U_(p)/U_(c) where b_(p) and b_(c) arethe thicknesses of the purge and separation chambers respectively. Theport 40 shown in FIG. 3 confines the sample stream to the center regionof the chamber. This configuration avoids the adverse effects ofelectroosmotic flows at the end walls. The evacuated glass side walls 39preclude heat transfer in the lateral direction as FIG. 2 shows. Thiscondition eliminates any variance in this direction so that scale-up ofthe sample stream width is unlimited.

38. Referring to FIG. 2, the focusing flow V_(o) causes a temperaturegradient in the transverse direction as it brings cooler flow from thepurge chamber 5 into the separation chamber 1. Also, referring to FIG.1, the purge flow which cools the separation chamber is heated as itmoves through the chamber giving rise to an axial temperature gradientin the separation chamber. The first gradient gives rise to a clockwisecirculation when the chamber is in the vertical orientation, while thesecond gradient gives rise to a similar clockwise circulation when thechamber is in the horizontal orientation. Mathematical models show thatthe circulations are attenuated to a much greater extent by the chamberwalls when the chamber is in the horizontal orientation. However, byutilizing internal injection and collection ports 12, 13 the effect ofthe circulations is eliminated since the disturbance flows occur in thefront of port 12 and behind port 13 and hence does not affect theseparation. Thus there is no effect of chamber orientation on thermalconvection disturbances with internal injection and collection ports.The above configuration is adaptable to voltage gradients up to about100 V/cm. However, if higher voltage gradients are preferred, somemodification should be considered.

39. The high voltage (second) configuration is characterized by a verythin (transverse thickness) separation chamber and the elimination ofthe internal injection and collection ports 12 and 13. Sample isinjected through port 21 and collected through port 24. Hence, samplefills the entire separation chamber with no buffer zones whichcharacterize the low voltage configurations. The smaller heated volumesof the separation and purge chambers limit the chamber temperature athigh electric fields. A significant advantage of having only sample inthe separation chamber is the homogeneity of the electrical conductivityin the field direction. Since Joule heating and electrohydrodynamicsboth vary as the electric field squared, and since the adverse effectsof both are dependent on electric field gradients, it is important toeliminate these gradients if high voltage gradients are to besuccessfully used. The high voltage configuration must be operated inthe horizontal orientation. While lateral gradients are controlled bythe insulated ends 39, transverse temperature gradients can besignificant at high voltage gradients and are exacerbated by the coolfocusing cross-flow from purge chamber 5 into the separation chamber.The focusing flow V_(o) produces a gradient of increasing temperaturefrom purge chamber 5 through the separation chamber to purge chamber 4.This gradient can give rise to significant circulation if the chamber isoperated in the vertical orientation aligned with gravity. If thechamber is operated in the horizontal orientation, this circulation issuppressed as our mathematical models of the chamber configuration haveshown. Since the chamber is cooled by the purge flows of velocity U_(p)emitting from the purge manifolds, an axial temperature gradient is alsodeveloped in the chamber, however, it is generally much smaller thanthat produced by the transverse gradient and is similarly suppressed byhorizontal operation.

40. II. Varying Voltage Gradient Configuration

41. The third configuration shown in FIGS. 4, 5 and 6 can be describedas a true focusing system. The significant features of this innovationare the use of a constant transverse flow and a varying transverseelectric field that is linear in the transverse coordinate. The electricfield can be described by:

E _(z) =B−Cz,

42. where z is perpendicular to the direction of the carrier bufferflow, B is the field at z=0 and C is a linear constant. Thecorresponding electrophoretic velocity of sample fraction i is:

v _(e)=μ_(i) E _(z) =μ _(i)(B−Cz)

43. Focusing is achieved by the imposition of a constant fluid flow ofspeed V₀ with a direction opposing v_(e). The velocity of a singleionized molecule of the i^(th) sample fraction is:

dz _(e) /dt=−V _(o)+μ_(i)(B−Cz)  (1)

44. where z_(e) is the electrophoretic displacement of the fraction inthe z-direction. Each fraction approaches a null position z_(i) givenby:

V _(o)=μ_(i)(B−Cz _(i))  (2)

45. Solving equation 1,

z _(e)=(z _(o) −z _(i))exp(−μ_(i) Ct)+z _(i)  (3)

46. where z_(o) is the initial ion position. For large t, the first termapproaches zero so that the final (focused) position z_(i) isindependent of the initial position, z_(o), and is the position obtainedfrom equation 2. The advantage of focusing is that each fraction movestoward a fixed position in the separation chamber. This is in contrastto zonal techniques where fractions move to relative positions withrespect to other fractions.

47. FIGS. 4 and 5 show a multi-chamber configuration similar to thefirst two configurations. The separation chamber 1 is much wider in thiscase in order to accommodate the multitude of injection 12 andcollection ports 13 necessary to handle the sample mobility spectrum ofinterest. The single electrodes of FIGS. 1 and 2 have been replaced withelectrode arrays 10 and 11. Each element can be impressed with aspecific voltage and is the mechanism by which the linear voltagegradient Ez is produced. For focusing, sample and buffer is injectedthrough the port array 12 with the separated fraction streams collectedat the exit port array 13. Flow occurs in the separation chamber 1 onlybetween the port arrays 12, 13. Flow stagnation regions 42 and 43respectively exist in front of port array 12 and in back of port array13. The ports 21 and 24 are only active initially during filling of thechamber. These stagnation regions serve both as electrical current andthe fluid flow return paths. FIG. 7 shows a simplified schematic of thechamber with a coordinate system to facilitate a description of theelectrode and flow field needed for focusing and which will justify theport and electrode modifications described above.

48. The total rectangular chamber is bounded by:

49. |x|<a; |y|<b and |z|<c, where y is the orthogonal axis to the x-zplane. The total chamber dimensions are double these values. It isseparated into three segments by screens 2, 3 at |z|= e.

50. The linear electric field gradient E_(z) is provided by electrodearrays 10 and 11 distributed along the line |z|=c and are kept at thefollowing voltages:

V=−{Ax+Bz+½C(x ² −z ²)}

51. where the constants A, B and C are independent and computercontrolled to optimize the separation. The purpose of these electrodesis to maintain this voltage V (which locally satisfies Laplace'sequation, ∇²V=0) throughout the separation chamber 1 and its fouradjacent segments 4, 5, 26, and 27. The insulating walls at |y|=b do notmodify this voltage distribution. The inflow and outflow ports 12, 13 at|x|=d will not modify the electric field distribution provided each portis electrically isolated and there are adequate gaps in the z-directionbetween the ports, as FIG. 6 shows. The corresponding electric fieldcomponents are:

E _(x) =A+Cx  (4)

E _(z) B−Cz  (5)

52. Equation 5 produces focusing while equation 4 causes a linearvariation in the axial ion motion affecting residence time which can beused to promote sample concentration.

53. To see how equations 4 and 5 satisfy ∇²V=0, a numerical solution isdone for the region bounded by |z|=c and |x|=a. The boundary conditionat |z|=c is

V=−½C(x ² − ²)

54. where A=0 and B=0 so that only the C terms are considered. The fieldlines for this solution are shown in FIG. 8. This computation was donefor a/c=20; note that the z-dimension is exaggerated in the display.Note that the field lines indicate that equations 4 and 5 are goodapproximations except near the walls at |x|=a and |z|=c. This region ofconformity is bounded by |z|=e and |x|=d and is the separation chamber.The stagnation regions 42, 43 also provide a return path for the currentconverging near the walls at |x|=a. These return paths assure that

E _(z) =B−Cz

55. in the separation chamber 1. In order to improve resolution, samplefrom the collection ports 13 can be recycled back into theircorresponding entry ports 12 to form a recycling mode of operation. Thismode will be a batch type for purposes of sample collection.

56. The preferred embodiment for detection and collection of the focusedsample streams is to use a UV scan in front of the collection ports 13to establish the position of the fractions and generate a conventionalhistogram for the fractions observed in the mobility spectrum currentlyunder observation. One port should be used to collect a fraction ofinterest by positioning that fraction over the collection port bymanipulation of either V₀ or E₀. This process is most easily handledthrough the use of computer control. Other fraction spectra can beobserved and/or collected by further manipulation of V₀ or E₀.

57. As one of ordinary skill in this art would recognize, the abovedescriptive embodiments are only exemplary of the present invention, andthere are numerous modifications and alternative modes that would fallwithin the scope of the invention, which is set forth in the claimsappended hereto.

What is claimed is:
 1. A method for separation and collection of atleast one component from a mixture of components comprising the stepsof: a. providing an apparatus comprising a separation chamber and aplurality of purge chambers, and establishing a first buffer flow in theseparation chamber in the axial direction, said first buffer flow havinga first flow rate; b. establishing a second buffer flow in each of atleast two of said purge chambers in the axial direction, said secondbuffer flow having a second flow rate, said second buffer fluid flowhaving a second flow rate higher than that of the first flow rate; c.establishing a third buffer flow in a plurality of purge chambersaxially adjacent to said separation chamber and separated from theseparation chamber by metal-coated glass screens; d. establishing afourth buffer flow into one of the purge chambers laterally through theseparation chamber, then into and out of a second purge chamber toprovide a uniform focusing fluid velocity in the separation chamber; e.introducing the mixture of components directly into the separationchamber flow entrance or through at least one injection port located inthe separation chamber interior; f. applying an electrical potentialtransversely across the separation chamber in the form of a constantvoltage gradient or a linearly varying voltage gradient to impartelectrophoretic velocity to the fractional components in the separationchamber in the transverse direction perpendicular to the first bufferflow direction and parallel to the third buffer flow direction; and g.withdrawing at least one separated sample component.
 2. A methodaccording to claim 1 wherein the separated sample is withdrawn at theflow exit of the separation chamber.
 3. A method according to claim 1wherein the separated sample is withdrawn through a single collectionport or from each of a plurality of collection ports.
 4. A methodaccording to claim 1 wherein the sample is injected with the carrierflow at the flow entrance of the separation chamber and is acted on bythe combined influences of a constant electric field and said third flowtransversely across the separation chamber.
 5. A method according toclaim 1 wherein one sample component is maintained in the separationchamber while extraneous components are discarded through the purgechambers.
 6. A method according to claim 1 wherein the third buffer flowis adjusted to provide a transversely varying cross-flow velocity whichallows any selected sample component to be either analyzed or collected.7. A method according to claim 6 wherein the selected sample componentis collected at the flow exit of the separation chamber.
 8. A methodaccording to claim 1 wherein the sample is injected through a singleinjection port into the separation chamber while carrier buffer entersthe separation chamber at the fluid flow entrance of the separationchamber.
 9. A method according to claim 8 wherein the sample is acted onby the combined influences of a constant electric field and said thirdbuffer flow transversely across the separation chamber.
 10. A methodaccording to claim 9 wherein one sample fraction is maintained in theseparation chamber and arrives at a single collection port in theseparation chamber while extraneous components are either discardedthrough the said purge chambers or flow around the collection port andout of the separation chamber at the carrier buffer flow exit.
 11. Amethod according to claim 1 wherein the said third buffer flow isadjusted to provide a transversely varying cross-flow velocity whichallows any selected sample component to be either analyzed or collectedat a single collection port.
 12. A method according to claim 1 whereinthe fraction spectrum of a sample may be analyzed or collected byvarying the flow of a pump in a linear variation to present atime-dependent histogram.
 13. A method according to claim 1 wherein thesample and buffer solution is injected through a plurality of inputports located at the entrance region of the separation chamber.
 14. Amethod according to claim 13 wherein the sample fractions are acted onby the combined influences of a linearly varying electric field and athird buffer flow transversely across the separation chamber.
 15. Amethod according to claim 1 wherein the sample fractions migrate towardset transverse positions near the exit end of the separation chamber.16. A method according to claim 15 wherein the sample fractions may bescanned in the exit region of the separation chamber by a UV laser anddetector system with sample fractions being collected in a single ormultiple set of collection ports.
 17. A method according to claim 16wherein the sample fraction spectrum in the separation chamber is fixedby the transverse chamber thickness with the remainder of the spectrumbeing diverted through screens into and out of the purge chambers.
 18. Amethod according to claim 15 wherein the sample fraction spectrum beingviewed may be changed by varying the third flow rate of focusing fluidvelocity.
 19. A method according to claim 17 wherein the sample fractionspectrum being viewed may be collected in singular or multiplecollection ports by varying the third flow rate of focusing fluidvelocity.
 20. A method according to claim 3 wherein the sample enteringcollection ports may be recycled back to the corresponding sample entryports to form a recycling process which can improve resolution byincreasing sample residence time.